Nitric oxide synthase (NOS) catalyzes the oxygenation of arginine to nitric oxide (NO) and citrulline. The enzyme is unique in requiring 5 factors for activity, including tetrahydrobiopterin (BH4). In addition to NO synthesis, neuronal NOS catalyzes the uncoupled activation of oxygen (especially when BH4 or arginine are not saturating) to form the toxic oxidants, superoxide, peroxide and peroxynitrite. One of the many roles proposed for NO is that it acts as a neurotransmitter in the brain, and appears to be involved in the physiology of learning and memory. Clarification of the action of BH4 in nitric oxide formation is therefore important in understanding neurotransmission and some of its abnormal aspects. It is well established that BH4 has marked physical effects on NOS, including the promotion of arginine binding and stabilization of the active dimeric state. However, little is known of the chemical reactivity of NOS bound BH4 and the possibility, for example, that it may be a stoichiometric redox reactant in the formation of NO. We have recently observed that during normal multiple turnovers of NOS, NOS- bound BH4 does not remain in a static state, but cycles between the reduced and oxidized forms. BH4 oxidation was increased in the presence of the substrate, arginine. These combined observations suggest that BH4 may be acting as an electron donor in the oxygenation of arginine to form NO. We have followed up on this proposal by examining whether NOS bound BH4 is converted to the carbinolamine (4-alpha-hydroxy tetrahydrobiopterin) during NO formation. The carbinolamine (6- and 7-isomers) are known to be formed only as products of the reaction in which BH4 acts as a stoichiometric reactant in the oxygenations catalyzed by the aromatic amino acid hydroxylases. Any such formation of the carbinolamine by NOS would therefore provide definitive evidence that a similar stoichiometric role of BH4 also operates for this enzyme. We are currently examining whether the carbinolamine is formed during catalysis by NOS, using sensitive and specific detection methods developed in our laboratory. - nitric oxide synthase, N-hydroxyarginine, tetrahydrobiopterin, superoxide, peroxynitrite, carbinolamine